The present invention relates to a method for analyzing the primary structure of a protein or a peptide.
In order to determine an amino acid sequence of a protein or a peptide from a carboxy terminus thereof (hereinafter sometimes referred to as a C-terminal), a method, such as that shown in FIG. 3 is used, comprising steps of allowing a protein or a peptide to react with carboxypeptidase, collecting portions of the reaction mixture with the passage of time, analyzing the reaction mixture by an amino acid analyzer, and analyzing the liberated amino acid (Seikagakujikkenkouza Vol.1, Tanpakushitunokagaku II, edited by Nihonseikagakukai, p203-211, 1976).
And, a method for analyzing the mass of a C-terminal truncated protein or a C-terminal truncated peptide by analyzing the reaction mixture using a mass spectrograph is reported (A. Tsugita, R. van den Broek, M. Pyzybylski, FEBS. Lett. 137, 19(1982)). Also, as an example of the chemical methods, a method for analyzing a sequence comprising repeating a series of operations consisting of activating the C-terminal with acetic anhydride, binding trimethylsilylisothiocyanate, and cleaving with hydrochloric acid is reported as shown in FIG. 4 (D. H. Hawke, H-. W. Lahm, J. E. Shively, C. W. Todd, Anal. Biochem. 166, 298(1987)).
The conventional method using carboxypeptidase has difficulty in accurate determination by cleavage at undesirable peptide bonds derived from the diversity of substrate specificity and activity of the enzyme depending upon the C-terminal amino acid or an adjacent amino acid thereof, and from the contamination of the other protease. Also this method is not suitable for microanalysis, since the contamination by the mixture therein of amino acid and peptide occurs because of the reasons that the enzyme has an autolytic property and is unsatisfactorily purified sometimes.
Further, the other chemical method is not sufficiently made to a practical use.